Neurosteroid analogues produce general anesthesia by binding to GABA-A receptors and enhancing neuronal inhibition in the brain. The number of binding sites for neurosteroids on GABA-A receptors and their molecular localization has been incompletely defined and is based on indirect methods. Neurosteroids are also endogenous mediators of brain development and function whose sites of action remain undefined. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on GABA-A receptors. Novel click chemistry-tagged, neurosteroid analogue photolabeling reagents will be developed and used in conjunction with isotopically tagged, click chemistry bifunctional linkers, to purify photolabeled proteins and peptides, providing the sensitivity and specificity to use high resolution mass spectrometry to detect and sequence low-abundance photolabeled peptides in biological samples. These techniques will also be applied as a discovery tool to identify additional proteins in brain that have neurosteroid binding sites. The Project has three specific aims: In Aim 1, novel neurosteroid analogue photolabeling reagents containing an alkyne group and isotopically-tagged, bifunctional linker molecules containing both an azide group and an affinity tag will be synthesized for use in click chemistry-based visualization and purification of photolabeled proteins and peptides (Aims 2 and 3). The isotopic tag will provide a signature for specific mass spectrometric detection of photolabeled peptides. Neurosteroid analogues will be synthesized that differ in stereochemistry, mechanism of photolabeling and the location of the diazirine photolabeling moiety on the neurosteroid backbone. In Aim 2 the number and structure of the neurosteroid binding sites on a defined GABA-A receptor (alpha1beta3) will be assessed by photolabeling with the neurosteroid analogues prepared in Aim 1. Incorporating this photolabeling data in a molecular model of the receptor will define the geometry of the binding site(s). Aim 2 will also quantitatively analyze which GABA-A receptor subunits are photolabeled by neurosteroids in native brain tissue. In Aim 3, photolabeling will be used to identify additional proteins that have neurosteroid binding sites. Whole brain homogenates will be photolabeled with neurosteroid analogue photolabeling reagents and the labeled proteins will be identified either: 1) by using click chemistry-based enrichment of photolabeled proteins in conjunction with global proteomic methods or; 2) by using bifunctional fluorescent linkers to track the photolabeled proteins through electrophoretic separation, and then identifying the proteins using gel-based mass spectrometry. The proposed work is expected to identify the sites of neurosteroid binding on GABA-A receptors and to discover new neurosteroid binding proteins. These data will provide a structural template for future synthetic chemistry and suggest novel mechanisms of neurosteroid action and potential pharmacological targets.